Fertilization is an ancient process whose essence is an interaction between two single cells that culminates in creation of one cell. A cell of one sex - typically driven by a flagellum - - contacts a cell of the opposite sex, one or both are activate, and then they fuse. We still lack fundamental information about the molecules and mechanisms that accomplish metazoan gamete fusion and a candidate vertebrate gamete fusion protein remains unidentified. The unicellular, biflagellated alga Chlamydomonas is a unique entry point into studying and understanding the evolutionary development of fertilization. In Chlamydomonas, recognition and adhesion between the highly motile plus and minus gametes bring them together and trigger both to erect cell membrane sites specialized for fusion, the mating structures. Attachment and bilayer merger of the two membranes are genetically distinguishable. Pre-fusion attachment requires interaction of FUS1 on the plus mating structure with MAR1 on the minus mating structure. Bilayer merger depends on the broadly conserved HAP2 protein on the minus mating structure. These requirements for two sets of proteins and for HAP2 in fusion have been confirmed in protists and higher plants and are implicated in some metazoans. Plasmodium HAP2 has become a target for a transmission-blocking malaria vaccine. New studies of functional domains of HAP2 have identified a motif in the cytoplasmic domain essential for targeting to the site of fusion and a conserved cytoplasmic motif that regulates the fusion function of the protein. A new comprehensive analysis of the Chlamydomonas transcriptome has identified a set of genes that are uniquely expressed in gametes and revealed a previously unrecognized family of nuclear envelope proteins essential for zygotic pronuclear fusion across domains of life. Biochemical studies show that MAR1 and HAP2 interact with each within the membrane of the mating structure membrane of minus gametes, and that FUS1, MAR1, and HAP2 are in a common complex during the membrane fusion reaction. More information is available about the cellular and molecular underpinning of gamete fusion in Chlamydomonas than in any other organism. Understanding, for at least one organism, the molecular events that occur during the gamete membrane fusion reaction will establish a framework for dissecting fundamental principles of gamete fusion in other organisms and will lead to development of vaccines to block fertilization of human parasitic pathogens, to development of contraceptives, and to treatments for infertility. The objective of the research proposed here is to test the model that HAP2 functions as a fusion protein during the membrane fusion reaction. The aims are to identify domains of HAP2 required for fusion and investigate whether the HAP2-MAR1 interaction is essential for the fusion function of HAP2; to examine the unique properties of the specialized FUS1-MAR1-dependent adhesion junction that contribute to bilayer fusion; and to identify the minimal set of proteins required to reconstitute the membrane fusion reaction in a heterologous system.